Novel metastasis suppressor gene on human chromosome 8

ABSTRACT

An isolated or purified nucleic acid molecule encoding the metastasis suppressor gene, Tey 1, located at p21-p12 on human chromosome 8. The invention further includes methods of treating cancer comprising administering a nucleic acid molecule encoding Tey 1 or administering an isolated Tey 1 polypeptide. The invention further includes methods of diagnosing or prognosticating cancer in a mammal comprising assaying for the level of Tey 1.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a metastasis suppressor gene located onchromosome 8 in humans and related vectors, host cells, polypeptides,compositions and methods of diagnosis, prognosis and treatment ofcancer.

BACKGROUND OF THE INVENTION

The American Cancer Society estimates the lifetime risk that anindividual will develop cancer is 1 in 2 for men and 1 in 3 for women.The development of cancer, while still not completely understood, can beenhanced as a result of a variety of risk factors. For example, exposureto environmental factors (e.g., tobacco smoke) might triggermodifications in certain genes, thereby initiating cancer development.Alternatively, these genetic modifications may not require an exposureto environmental factors to become abnormal. Indeed, certain mutations(e.g., deletions, substitutions, etc.) can be inherited from generationto generation, thereby imparting an individual with a geneticpredisposition to develop cancer.

Currently, the survival rates for many cancers are on the rise. Onereason for this success is improvement in the detection of cancer at astage at which treatment can be effective. Indeed, it has been notedthat one of the most effective means to survive cancer is to detect itspresence as early as possible. According to the American Cancer Society,the relative survival rate for many cancers would increase by about 15%if individuals participated in regular cancer screenings. Therefore, itis becoming increasingly useful to develop novel diagnostic tools todetect the cancer either before it develops or at an as early stage ofdevelopment as possible.

One popular way of detecting cancer early is to analyze the geneticmakeup of an individual to detect the presence or expression levels of amarker gene(s) related to the cancer. For example, there are variousdiagnostic methods that analyze a certain gene or a pattern of genes todetect cancers of the breast, tongue, mouth, colon, rectum, cervix,prostate, testis, and skin.

Prostate cancer is the most common non-cutaneous malignancy diagnosed inmen in the United States, accounting for over 40,000 deaths annually(Parker et al., J. Clin. Cancer 46: 5 (1996)). While methods for earlydetection and treatment of prostate cancer have been forthcoming, thereis an obvious need for improvement in this area. Therefore, thediscovery of gene mutations which are good indicators of cancer, andmore particularly prostate cancer, would be a tremendous step towardsunderstanding the mechanisms underlying cancer and could offer adramatic improvement in the ability of scientists to detect cancer andeven to predict an individual's susceptibility to a particular type ofcancer.

Much research has, in fact, been centered around establishing a geneticlink to prostate cancer and studies have identified many recurringgenetic changes associated with prostate cancer. These genetic changesinclude DNA hypermethylation, allelic loss, aneuploidy, aneusomy,various point mutations, and changes in protein expression level (e.g.,E-cadherin/alpha-catenin). Researchers have also discovered losses andduplications in particular chromosomes or chromosome arms which areassociated with prostate cancer (U.S. Pat. No. 5,925,519; Visakorpi,Ann. Chirur. Gynaec. 88:11-16 (1999)). In particular, losses ofchromosomes 6q, 8p, 10q, 13q and 16q, and duplications of 7, 8q and Xqhave be an associated with prostate cancer. Moreover, researchers haveperformed genetic epidemiological studies of affected populations andhave identified various putative prostate cancer susceptibility loci,indicating that there is significant genetic heterogeneity in prostatecancer. These include Xq27-q28 (Xu et al., Nat. Genet. 20:175-179(1998)) and 1q42-q43 (Gibbs et al., Am. J. Hum. Genet. 64:1087-1095(1999); Berthon et al., Am. J. Hum. Genet. 62:1416-1424 (1998)).

One such potential prostate cancer susceptibility locus is the 1 q24-q31locus (flanked by D1S2883 and D1S422), which has been designated as HPC1(due to its putative link to hereditary prostate cancer (HPC)). ThisHPC1 locus was identified in a genome-wide scan of families at high riskfor prostate cancer (Smith et al., Science 274:1371-1374 (1996)). TheHPC1 locus has been controversial, however, due to the fact thatresearchers have had difficulty duplicating the results of Smith et al.(De la Chapelle et al., Curr. Opin. Genet. Dev. 8:298-303 (1998)). Infact, some groups of researchers have found no linkage of the HPC1 locusto hereditary prostate cancer (Eeles et al., Am. J. Hum. Genet.62:653-658 (1998); Thibodeau et al., Am. J. Hum. Genet. 61(suppl.):1733(1997); McIndoe et al., Am. J. Hum. Genet. 61:347-353 (1997)), whileothers have found linkage in a very small fraction of high-risk prostatecancer families (Schleutker et al., Am. J. Hum. Genet. 61(suppl.):1711(1997)). Further support for the linkage between the HPC1 locus andhereditary prostate cancer was revealed, however, via a combinedConsortium analysis of 6 markers in the HPC1 region in 772 familiessegregating hereditary prostate cancer (see Xu et al., Am. J. Hum.Genet. 66:945-957 (2000)). In this regard, research findings concerningthe HPC1 locus and its potential link to prostate cancer have beenpromising, but often nonconforming.

There also have been numerous reports of allelic loss of the p arm ofchromosome 8 associated with prostate cancers (as high as 65% ofprostate carcinomas) (see, e.g., Bookstein et al., U.S. Pat. No.6,043,088). Numerous reports of genes associated with cancer and, morespecifically, prostate cancer (see, e.g., An et al., U.S. Pat. Nos.5,882,864; 5,972,615; 6,156,515; 6,171,796; and 6,218,529), onchromosome 8 (see, e.g., Ichikawa et al., Cancer Research 54: 2299-2302(1994), and Kuramochi et al., The Prostate 31: 14-20 (1997)), in rehuman chromosome 8; Nihei et al., Genes, Chromosomes & Cancer 17:260-268 (1996), and Ichikawa et al., Asian J. of Andrology 2(3): 167-171(2000), in re metastasis suppressor gene at p21-p12 on chromosome 8;Ichikawa et al., The Prostate Supplement 6: 31-35 (1996), in remetastasis suppressor gene at 8p23-q12; Nihei et al., Proc. 90th Ann.Mtg. of the Amer. Assoc. Cancer Research 40: 105 (Abstract No. 699)(March 1999), in re metastasis suppressor gene at D8S131-D8S339 on the parm of human chromosome 8; Sunwoo et al., Oncogene 18: 2651-2655 (1999),in re tumor suppressor at D8S264-D8S1788; Trapman et al., CancerResearch 54: 6061-6064 (1994), in re tumor suppressor gene atD8S87-D8S133; Konig et al., Urol. Res. 27(1): 3-8 (1999), in re tumorsuppressor gene at 8p21 (see, also, Kagan et al., Oncogene 11: 2121-2126(1995), and He et al., Genomics 43: 69-77 (1997)); Bookstein et al.(U.S. Pat. No. 6,043,088) in re prostate/colon tumor suppressor geneproduct (PTSG protein) from p22 region of chromosome 8 (see, also, Levyet al., Genes, Chromsomes & Cancer 24: 4247 (1999), and Kagan et al.,supra, in re homozygous deletions in this region); Cohen et al.,International Patent Application No. WO 99/32644 in re PG1 gene from p23region of chromosome 8; Wang et al., Genomics 60: 1-11 (1999), in retumor suppressor gene at 8p22-p23; and Oba et al., Cancer Genet.Cytogenet. 124: 20-26 (2001), in re two putative tumor suppressor genesat p21.1-p21.2 and p22-p21.3 (see, also, Suzuki et al., Genes,Chromsomes & Cancer 13; 168-174 (1995), on chromosome 8).

8p11 has been found to be a recurrent chromosomal breakpoint in prostatecancer cell lines (Pan et al., Genes, Chromosomes & Cancer 30: 187-195(2001). It also has been reported that loss of 8p sequences may resultfrom complex structural rearrangements involving chromosome 8, whichsometimes includes i(8q) chromosome formation (Macoska et al., CancerResearch 55: 5390-5395 (1995), and Cancer Genet. Cytogenet. 120: 50-57(2000)). Genetic changes at 8q in clinically organ-confined prostatecancer also have been noted (Fu et al., Urology 56: 880-885 (2000)).Differential expression of the gene GC84 at 8q11 has been associatedwith the progression of prostate cancer (Chang et al., Int. J. Cancer83: 506-511 (1999)). 8p22 loss with 8c gain has been associated withpoor outcome in prostate cancer (Macoska et al., Urology 55: 776-782(2000)); see, also, Arbieva et al., Genome Research 10: 244-257 (2000)).Loss of 8p23 and 8q12-13 has been found to be associated with humanprostate cancer (Perinchery et al., Intel. J. Oncology 14: 495-500(1999)). Gene amplification in 8q24 has been found to be associated withhuman prostate cancer (McGill et al., International Patent ApplicationNo. WO 96/20288). Mutations in the FEZ1 gene at 8p22 have been found tobe associated with primary esophageal cancers and in a prostate cancercell line (Ishii et al., PNAS USA 96: 3928-3933 (March 1999)).

The use of various gene sequences in the diagnosis and prognosis ofcancer, specifically prostate cancer, also has been disclosed (see,e.g., An et al., supra; Russell et al., U.S. Pat. No. 5,861,248; Jenkinset al., European Patent Application EP 1 048 740; Bachner et al., U.S.Pat. No. 6,140,049; Ross et al., U.S. Pat. No. 5,994,071; and Jensen etal., U.S. Pat. No. 5,925,519).

Genes and gene products, which can be shown to have a strong associationwith cancer, such as prostate cancer, need to be identified. Such genesand gene products would lead directly to early, sensitive and accuratemethods for detecting cancer or a predisposition to cancer in a mammal.Moreover, such methods would enable clinicians to monitor the onset andprogression of cancer in an individual with greater sensitivity andaccuracy, as well as the response of an individual to a particulartreatment. The present invention provides such a gene and gene products,as well as related vectors, host cells, compositions and methods of usein the diagnosis, prognosis and treatment of cancer, in particularprostate cancer.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated or purified nucleic acidmolecule consisting essentially of a nucleotide sequence encoding themetastasis suppressor gene located at p21-p12 on chromosome 8 of ahuman, which has been named Tenni Yokusei 1 (Tey 1), or a fragmentthereof comprising at least 455 contiguous nucleotides.

The present invention also provides an isolated or purified nucleic acidmolecule consisting essentially of a nucleotide sequence encoding avariant Tey 1 or a fragment thereof comprising at least 455 contiguousnucleotides. The variant Tey 1 comprises one or more insertions,deletions, substitutions, and/or inversions and does not differfunctionally from the corresponding unmodified Tey 1.

Still also provided by the present invention is an isolated or purifiednucleic acid molecule consisting essentially of a nucleotide sequencethat is complementary to a nucleotide sequence encoding Tey 1 or afragment thereof comprising at least 455 contiguous nucleotides.

Thus, the present invention also provides an isolated or purifiednucleic acid molecule consisting essentially of a nucleotide sequencethat is complementary to a nucleotide sequence encoding a variant Tey 1.

In view of the above, the present invention further provides a vectorcomprising an above-described isolated or purified nucleic acidmolecule. When the isolated or purified nucleic acid molecule consistsessentially of a nucleotide sequence encoding Tey 1 or a variantthereof, the isolated or purified nucleic acid molecule is optionallypart of an encoded fusion protein.

Also in view of the above, the present invention provides a cellcomprising and expressing an above-described isolated or purifiednucleic acid molecule, optionally in the form of a vector.

An isolated or purified polypeptide molecule consisting essentially ofan amino acid sequence encoding Tey 1, a variant Tey 1, or at least 6contiguous amino acids of either of the foregoing, which is optionallyglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated or converted into an acid addition salt, is also provided bythe present invention. Thus, a conjugate or fusion protein comprisingthe isolated or purified polypeptide molecule or variant thereof and atherapeutically or prophylactically active agent is also provided as isa composition comprising the isolated or purified polypeptide molecule,optionally in the form of a conjugate or a fusion protein comprising atherapeutically or prophylactically active agent, and an excipient or anadjuvant.

In view of the above, a method of treating cancer prophylactically ortherapeutically in a mammal is also provided. The method comprisesadministering to the mammal an effective amount of (a) an isolated orpurified nucleic acid molecule encoding Tey 1, optionally in the form ofa vector, or (b) an isolated or purified Tey 1 polypeptide, optionallyin the form of a conjugate or fusion protein, whereupon the mammal istreated for the cancer prophylactically or therapeutically. Preferably,the cancer is prostate cancer.

Also in view of the above, a method of diagnosing cancer in a mammal isprovided. The method comprises (a) obtaining a test sample from themammal, and (b) assaying the test sample for the level of Tey 1, whereina decrease in the level of Tey 1 in the test sample as compared to thelevel of Tey 1 in a control sample is diagnostic for the cancer.

A method of prognosticating cancer in a mammal is also provided. Themethod comprises (a) obtaining a test sample from the mammal, and (b)assaying the test sample for the level of Tey 1, wherein an increase inthe level of Tey 1 over time is indicative of a positive prognosis and adecrease in the level of Tey 1 over time is indicative of a negativeprognosis. The method of prognosticating can be used to assess theefficacy of treatment of the cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the nucleotide sequence (SEQ ID NO: 1) of the cDNA of Tey 1,which is read 5′ to 3′ from top to bottom and left to right.

FIG. 2 is the nucleotide sequence (SEQ ID NO: 2) of an alternativelyspliced cDNA of Tey 1, which is read 5′ to 3′ from top to bottom andleft to right.

FIG. 3 is the deduced amino acid sequence (SEQ ID NO: 3) of thepolypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ IDNO: 2, which is read N-terminus to C-terminus from top to bottom andleft to right.

FIG. 4 is the nucleotide sequence (SEQ ID NO: 4) of an alternativelyspliced cDNA of Tey 1, which is read 5′ to 3′ from top to bottom andleft to right.

FIG. 5 is the deduced amino acid sequence (SEQ ID NO: 5) of thepolypeptide encoded by the nucleotide sequence of SEQ ID NO: 4, which isread N-terminus to C-terminus from top to bottom and left to right.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated or purified nucleic acidmolecule consisting essentially of a nucleotide sequence encoding Tey 1or a fragment thereof comprising at least 455 (or at least 475, 500, 550or 600 or more) contiguous nucleotides.

By “isolated” is meant the removal of a nucleic acid from its naturalenvironment. By “purified” is meant that a given nucleic acid, whetherone that has been removed from nature (including genomic DNA and mRNA)or synthesized (including cDNA) and/or amplified under laboratoryconditions, has been increased in purity, wherein “purity” is a relativeterm, not “absolute purity.” “Nucleic acid molecule” is intended toencompass a polymer of DNA or RNA, i.e., a polynucleotide, which can besingle-stranded or double-stranded and which can contain non-natural oraltered nucleotides. Desirably, the isolated or purified nucleic acidmolecule does not contain any introns or portions thereof.

Preferably, the isolated or purified nucleic acid molecule consistsessentially of a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 3 or 5, (ii) consists essentially of the nucleotide sequenceof SEQ ID NO: 1, 2 or 4 or a fragment thereof comprising at least 455contiguous nucleotides, (iii) hybridizes under low stringency conditionsto an isolated or purified nucleic acid molecule consisting essentiallyof the nucleotide sequence that is complementary to SEQ ID NO: 1, 2 or 4or a fragment thereof comprising at least 455 (or at least 475, 500, 550or 600 or more) contiguous nucleotides, or (iv) shares 50% (or 55%, 60%,65%, 70%, 75% or 80% or more) or more identity with SEQ ID NO: 1, 2 or4.

Also provided is an isolated or purified nucleic acid moleculeconsisting essentially of a nucleotide sequence encoding a variant Tey 1or a fragment thereof comprising at least 455 (or at least 475, 500, 550or 600 or more) contiguous nucleotides. The variant comprises one ormore insertions, deletions, substitutions, and/or inversions. Desirably,the variant Tey 1 does not differ functionally from the correspondingunmodified Tey 1, such as that comprising SEQ ID NO: 3 or 5. Preferably,the variant Tey 1 is able to suppress metastasis of a highly metastaticprostatic tumor cell line in vivo at least about 75%, more preferably atleast about 90% as well as the unmodified Tey 1 comprising SEQ ID NO: 3or 5 as determined by in vivo assay. The manner in which the assay iscarried out is not critical and can be conducted in accordance withmethods known in the art. Preferably, the one or more substitution(s)results in the substitution of an amino acid of the encoded Tey 1 withanother amino acid of equivalent mass, structure and/or charge.

The present invention also provides an isolated or purified nucleic acidmolecule consisting essentially of a nucleotide sequence that iscomplementary to a nucleotide sequence encoding Tey 1 or a fragmentthereof comprising at least 455 (or at least 475, 500, 550 or 600 ormore) contiguous nucleotides. Such an isolated or purified nucleic acidmolecule preferably (i) is complementary to a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 3 or 5, (ii) iscomplementary to the nucleotide sequence of SEQ ID NO: 1, 2 or 4 or afragment thereof comprising at least 455 contiguous nucleotides, (iii)hybridizes under low stringency conditions to an isolated or purifiednucleic acid molecule consisting essentially of SEQ ID NO: 1, 2 or 4 ora fragment thereof comprising at least 455 contiguous nucleotides, or(iv) shares 50% (or 55%, 60%, 65%, 70%, 75% or 80% or more) or moreidentity with the nucleotide sequence that is complementary to SEQ IDNO: 1.

Also provided is an isolated or purified nucleic acid moleculeconsisting essentially of a nucleotide sequence that is complementary toa nucleotide sequence encoding a variant Tey 1 as described above.

With respect to the above, one of ordinary skill in the art knows how togenerate insertions, deletions, substitutions and/or inversions in agiven nucleic acid molecule. See, for example, the references citedherein under “Example.” With respect to the above isolated or purifiednucleic acid molecules, it is preferred that any such insertions,deletions, substitutions and/or inversions are introduced such that themetastasis suppressor activity is not compromised or is even enhanced.It is also preferred that the one or more substitution(s) result(s) inthe substitution of an amino acid with another amino acid of equivalentsize, shape and charge.

Also with respect to the above, “does not differ functionally from” isintended to mean that the variant Tey 1 has activity characteristic ofthe unmodified Tey 1. In other words, it can suppress metastasis of atumor, particularly a prostatic tumor. However, the variant Tey 1 can bemore or less active than the unmodified Tey 1 as desired in accordancewith the present invention.

An indication that polynucleotide sequences are substantially identicalis if two molecules selectively hybridize to each other under stringentconditions. The phrase “hybridizes to” refers to the selective bindingof a single-stranded nucleic acid probe to a single-stranded target DNAor RNA sequence of complementary sequence when the target sequence ispresent in a preparation of heterogeneous DNA and/or RNA. “Stringentconditions” are sequence-dependent and will be different in differentcircumstances. Generally, stringent conditions are selected to be about20° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe.

For example, under stringent conditions, as that term is understood byone skilled in the art, hybridization is preferably carried out using astandard hybridization buffer at a temperature ranging from about 50° C.to about 75° C., even more preferably from about 60° C. to about 70° C.,and optimally from about 65° C. to about 68° C. Alternately, formamidecan be included in the hybridization reaction, and the temperature ofhybridization can be reduced to preferably from about 35° C. to about45° C., even more preferably from about 40° C. to about 45° C., andoptimally to about 42° C. Desirably, formamide is included in thehybridization reaction at a concentration of from about 30% to about50%, preferably from about 35% to about 45%, and optimally at about 40%.Moreover, optionally, the hybridized sequences are washed (if necessaryto reduce non-specific binding) under relatively highly stringentconditions, as that term is understood by those skilled in the art. Forinstance, desirably, the hybridized sequences are washed one or moretimes using a solution comprising salt and detergent, preferably at atemperature of from about 50° C. to about 75° C., even more preferablyat from about 60° C. to about 70° C., and optimally from about 65° C. toabout 68° C. Preferably, a salt (e.g., such as sodium chloride) isincluded in the wash solution at a concentration of from about 0.01 M toabout 1.0 M. Optimally, a detergent (e.g., such as sodium dodecylsulfate) is also included at a concentration of from about 0.01% toabout 1.0%. The following is an example of highly stringent conditionsfor a Southern hybridization in aqueous buffers (no formamide) (Sambrookand Russell, Molecular Cloning, 3rd Ed. SCHL Press (2001)):

Hybridization Conditions:

-   -   6×SSC or 6×SSPE    -   5× Denhardt's Reagent    -   1% SDS    -   100 ug/ml salmon sperm DNA    -   hybridization at 65-68° C.

Washing Conditions:

-   -   0.1×SSC/0.1% SDS    -   washing at 65-68° C.        Exemplary moderately stringent conditions, which allow for 25%        mismatch, are as follows:

Hybridization Conditions:

-   -   5×SSC or 5×SSPE    -   5× Denhardt's Reagent    -   100 μg/ml salmon sperm DNA hybridization at 50° C.

Washing Conditions:

-   -   1×SSC/0.1% SDS    -   washing at 55° C.        Exemplary low stringency conditions, which allow for 50%        mismatch, are as follows:

Hybridization Conditions:

-   -   5×SSC or 5×SSPE    -   5× Denhardt's Reagent    -   100 μg/ml salmon sperm DNA    -   hybridization at 25° C.

Washing Conditions:

-   -   2×SSC/0.1% SDS    -   washing at 37° C.

In view of the above, “highly stringent conditions” allow for up toabout 20% mismatch, preferably up to about 15% mismatch, more preferablyup to about 10% mismatch, and most preferably less than about 5%mismatch, such as 4%, 3%, 2% or 1% mismatch. “At least moderatelystringent conditions” preferably allow for up to about 45% mismatch,more preferably up to about 35% mismatch, and most preferably up toabout 25% mismatch. “Low stringency conditions” preferably allow for upto 89% mismatch, more preferably up to about 70% mismatch, and mostpreferably up to about 50% mismatch. With respect to the precedingranges of mismatch, 1% mismatch corresponds to one degree decrease inthe melting temperature.

The above isolated or purified nucleic acid molecules also can becharacterized in terms of “percentage of sequence identity.” In thisregard, a given nucleic acid molecule as described above can be comparedto a nucleic acid molecule encoding a corresponding gene (i.e., thereference sequence) by optimally aligning the nucleic acid sequencesover a comparison window, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) as compared to the reference sequence, which does notcomprise additions or deletions, for optimal alignment of the twosequences. The percentage of sequence identity is calculated bydetermining the number of positions at which the identical nucleic acidbase occurs in both sequences, i.e., the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Optimal alignment ofsequences for comparison may be conducted by computerizedimplementations of known algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis., or BlastN and BlastXavailable from the National Center for Biotechnology Information,Bethesda, Md.), or by inspection. Sequences are typically compared usingBESTFIT or BlastN with default parameters.

“Significant sequence identity” means that preferably at least 45%, morepreferably at least 50%, and most preferably at least 55% (such as 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more) of the sequence of a givennucleic acid molecule is identical to a given reference sequence.Typically, two polypeptides are considered to have “substantial sequenceidentity” if at least 45%, preferably at least 60%, more preferably atleast 90%, and most preferably at least 95% (such as 96%, 97%, 98% or99%) of the amino acids of which the polypeptides are comprised areidentical to or represent conservative substitutions of the amino acidsof a given reference sequence.

One of ordinary skill in the art will appreciate, however, that twopolynucleotide sequences can be substantially different at the nucleicacid level, yet encode substantially similar, if not identical, aminoacid sequences, due to the degeneracy of the genetic code. The presentinvention is intended to encompass such polynucleotide sequences.

While the above-described nucleic acid molecules can be isolated orpurified, alternatively they can be synthesized. Methods of nucleic acidsynthesis are known in the art. See, e.g., the references cited hereinunder “Example.”

The above-described nucleic acid molecules can be used, in whole or inpart (i.e., as fragments or primers), to identify and isolate relatedgenes from humans (and other mammals) for use in the context of thepresent inventive methods using conventional means known in the art.See, for example, the references cited herein under “Example.” It willbe possible to identify highly related Tey 1 nucleic acids usingportions of the sequence given in SEQ ID NO:1, for example.

In view of the above, the present invention also provides a vectorcomprising an above-described isolated or purified nucleic acidmolecule, optionally as part of an encoded fusion protein. A nucleicacid molecule as described above can be cloned into any suitable vectorand can be used to transform or transfect any suitable host. Theselection of vectors and methods to construct them are commonly known topersons of ordinary skill in the art and are described in generaltechnical references (see, in general, “recombinant DNA Part D,” Methodsin Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)and the references cited herein under “Example”). Desirably, the vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant or animal) into which the vector isto be introduced, as appropriate and taking into consideration whetherthe vector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid sequence as described above or a portionthereof ligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from ColE1, 2mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

Suitable vectors include those designed for propagation and expansion orfor expression or both. A preferred cloning vector is selected from thegroup consisting of the pUC series, the pBluescript series (Stratagene,LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEXseries (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as % GT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI101, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clontech).

An expression vector can comprise a native or normative promoteroperably linked to an isolated or purified nucleic acid molecule asdescribed above. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theskill in the art. Similarly, the combining of a nucleic acid molecule asdescribed above with a promoter is also within the skill in the art.

Optionally, the isolated or purified nucleic acid molecule can be partof an encoded fusion protein. The generation of fusion proteins iswithin the ordinary skill in the art (see, e.g., references cited under“Example”) and can involve the use of restriction enzyme orrecombinational cloning techniques (see, e.g., Gateway™ (Invirogen,Carlsbad, Calif.). See, also, U.S. Pat. No. 5,314,995.

Also in view of the above, the present invention provides a host cellcomprising and expressing an isolated or purified nucleic acid molecule,optionally in the form of a vector, as described above. Examples of hostcells include, but are not limited to, a human cell, a human cell line,E. coli (e.g., E. coli TB-1, TG-2, DH5α, XL-Blue MRF' (Stratagene),SA2821 and Y1090), B. subtilis, P. aerugenosa, S. cerevisiae, N. crassa,insect cells (e.g., Sf9, Ea4) and others set forth herein below.

The present invention further provides an isolated or purifiedpolypeptide molecule consisting essentially of an amino acid sequenceencoding Tey 1 or at least 6 (or at least 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 or more) contiguous amino acids of Tey 1, which isoptionally glycosylated, amidated, carboxylated, phosphorylated,esterified, N-acylated or converted into an acid addition salt. Methodsof protein modification (e.g., glycosylation, amidation, carboxylation,phosphorylation, esterification, N-acylation, and conversion into acidaddition salts) are known in the art.

Also provided is an isolated or purified polypeptide molecule consistingessentially of an amino acid sequence encoding a variant Tey 1 or atleast 6 (or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more)contiguous amino acids of a variant Tey 1, which is optionallyglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, converted into an acid addition salt.

The polypeptide preferably comprises an amino end and a carboxyl end.The polypeptide can comprise D-amino acids, Lamino acids or a mixture ofD- and L-amino acids. The D-form of the amino acids, however, isparticularly preferred since a polypeptide comprised of D-amino acids isexpected to have a greater retention of its biological activity in vivo,given that the D-amino acids are not recognized by naturally occurringproteases.

The polypeptide can be prepared by any of a number of conventionaltechniques. The polypeptide can be isolated or purified from a naturallyoccurring source or from a recombinant source. Recombinant production ispreferred. For instance, in the case of recombinant polypeptides, a DNAfragment encoding a desired peptide can be subcloned into an appropriatevector using well-known molecular genetic techniques (see, e.g.,Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (ColdSpring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning: ALaboratory Manual. 2^(nd) ed. (Cold Spring Harbor Laboratory, 1989). Thefragment can be transcribed and the polypeptide subsequently translatedin vitro. Commercially available kits also can be employed (e.g., suchas manufactured by Clontech, Palo Alto, Calif.; Amersham PharmaciaBiotech Inc., Piscataway, N.J.; InVitrogen, Carlsbad, Calif., and thelike). The polymerase chain reaction optionally can be employed in themanipulation of nucleic acids.

Alterations of the native amino acid sequence to produce variantpolypeptides can be done by a variety of means known to those skilled inthe art. For instance, site-specific mutations can be introduced byligating into an expression vector a synthesized oligonucleotidecomprising the modified site. Alternately, oligonucleotide-directedsite-specific mutagenesis procedures can be used such as disclosed inWalder et al., Gene 42: 133 (1986); Bauer et al., Gene 37: 73 (1985);Craik, Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584and 4,737,462.

With respect to the above isolated or purified polypeptides, it ispreferred that any such insertions, deletions and/or substitutions areintroduced such that the metastasis suppressor activity is notcompromised or is even enhanced. It is also preferred that the one ormore substitution(s) result(s) in the substitution of an amino acid withanother amino acid of equivalent mass, structure and charge.

Any appropriate expression vector (e.g., as described in Pouwels et al.,Cloning Vectors: A Laboratory Manual (Elsevier, N.Y.: 1985)) andcorresponding suitable host can be employed for production ofrecombinant polypeptides. Expression hosts include, but are not limitedto, bacterial species within the genera Escherichia, Bacillus,Pseudonmonas, Salmonella, mammalian or insect host cell systemsincluding baculovirus systems (e.g., as described by Luckow et al.,Bio/Technology 6: 47 (1988)), and established cell lines such as theCOS-7, C127, 3T3, CHO, HeLa, BHK cell line, and the like. The ordinarilyskilled artisan is, of course, aware that the choice of expression hosthas ramifications for the type of polypeptide produced. For instance theglycosylation of polypeptides produced in yeast or mammalian cells(e.g., COS-7 cells) will differ from that of polypeptides produced inbacterial cells, such as Escherichia coli.

Alternately, the polypeptide (including the variant polypeptides) can besynthesized using standard peptide synthesizing techniques well-known tothose of ordinary skill in the art (e.g., as summarized in Bodanszky,Principles of Peptide Synthesis, (Springer-Verlag, Heidelberg: 1984)).In particular, the polypeptide can be synthesized using the procedure ofsolid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30: 705-739(1987); and U.S. Pat. No. 5,424,398). If desired, this can be done usingan automated peptide synthesizer. Removal of the t-butyloxycarbonyl(t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blockinggroups and separation of the polypeptide from the resin can beaccomplished by, for example, acid treatment at reduced temperature. Thepolypeptide-containing mixture can then be extracted, for instance, withdimethyl ether, to remove non-peptidic organic compounds, and thesynthesized polypeptide can be extracted from the resin powder (e.g.,with about 25% w/v acetic acid). Following the synthesis of thepolypeptide, further purification (e.g., using high performance liquidchromatography (HPLC)) optionally can be done in order to eliminate anyincomplete polypeptides or free amino acids. Amino acid and/or HPLCanalysis can be performed on the synthesized polypeptide to validate itsidentity. For other applications according to the invention, it may bepreferable to produce the polypeptide as part of a larger fusionprotein, such as by the methods described herein or other genetic means,or as part of a larger conjugate, such as through physical or chemicalconjugation, as known to those of ordinary skill in the art anddescribed herein.

If desired, the polypeptides of the invention (including variantpolypeptides) can be modified, for instance, by glycosylation,amidation, carboxylation, or phosphorylation, or by the creation of acidaddition salts, amides, esters, in particular C-terminal esters, andN-acyl derivatives of the polypeptides of the invention. Thepolypeptides also can be modified to create polypeptide derivatives byforming covalent or noncovalent complexes with other moieties inaccordance with methods known in the art. Covalently-bound complexes canbe prepared by linking the chemical moieties to functional groups on theside chains of amino acids comprising the polypeptides, or at the N- orC-terminus.

Thus, in this regard, the present invention also provides a fusionprotein and a conjugate comprising an above-described isolated orpurified polypeptide molecule or fragment thereof and a therapeuticallyor prophylactically active agent. “Prophylactically” as used herein doesnot necessarily mean prevention, although prevention is encompassed bythe term. Prophylactic activity also can include lesser effects, such asinhibition of the spread of cancer. Preferably, the active agent is ananti-cancer agent. Methods of conjugation are known in the art. Inaddition, conjugate kits are commercially available. For examples ofmethods of conjugation and conjugates see, e.g., Hermanson, G. T.,Bioconjugate Techniques 1996, Academic Press, San Diego, Calif.; U.S.Pat. Nos. 6,013,779; 6,274,552 and 6,080,725 and Ragupathi et al.,Glycoconjugate Journal 15: 217-221 (1998).

The present invention also provides a composition comprising anabove-described isolated or purified polypeptide molecule, optionally inthe form of a conjugate or a fusion protein comprising aprophylactically or therapeutically active agent, and an excipient or anadjuvant. Excipients and adjuvants are well-known in the art, and arereadily available. The choice of excipient/adjuvant will be determinedin part by the particular route of administration and whether a nucleicacid molecule or a polypeptide molecule (or conjugate or fusion proteinthereof) is being administered. Accordingly, there is a wide variety ofsuitable formulations for use in the context of the present invention,and the invention expressly provides a pharmaceutical composition thatcomprises an active agent of the invention and a pharmaceuticallyacceptable excipientladjuvant. The following methods andexcipients/adjuvants are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluent, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth. Pastilles can comprise the active ingredientin an inert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients/carriers as are known in the art.

An active agent of the present invention, either alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also canbe formulated as pharmaceuticals for non-pressured preparations such asin a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, active agents of the present invention can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. Formulations suitable for vaginaladministration can be presented as pessaries, tampons, creams, gels,pastes, foams, or spray formulas containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.Further suitable formulations are found in Remington's PharmaceuticalSciences, 17th ed., (Mack Publishing Company, Philadelphia, Pa.: 1985),and methods of drug delivery are reviewed in, for example, Langer,Science 249: 1527-1533 (1990).

In view of the above, the present invention provides a method oftreating cancer prophylactically or therapeutically in a mammal. Themethod comprises administering to the mammal an effective amount of (a)an isolated or purified nucleic acid molecule encoding Tey 1, optionallyin the form of a vector, or (b) an isolated or purified Tey 1polypeptide, optionally in the form of a conjugate or fusion protein,whereupon the mammal is treated for the cancer prophylactically ortherapeutically. Preferably, the cancer is prostate cancer.

The anti-cancer agent can be a chemotherapeutic agent, e.g., a polyamineor an analogue thereof. Examples of therapeutic polyamines include thoseset forth in U.S. Pat. Nos. 5,880,161, 5,541,230 and 5,962,533, Saab etal., J. Med. Chem. 36: 2998-3004 (1993), Bergeron et al., J. Med. Chem.37(21): 3464-3476 (1994), Casero et al., Cancer Chemother. Pharmacol 36:69-74 (1995), Bernacki et al., Clin. Cancer Res. 1: 847-857 (1995);Bergeron et al., J. Med. Chem. 40: 1475-1494 (1997); Gabrielson et al.,Clinical Cancer Res. 5: 1638-1641 (1999), and Bergeron et al., J. Med.Chem. 43: 224-235 (2000), which can be administered alone or incombination with other active agents, such as anti-cancer agents, e.g.,cis-diaminedichloroplatinum (II) and1,3-bis(2-chloroethyl)-1-nitrosourea.

Preferred routes of administration in the first embodiment of the methodof treating cancer include intratumoral and peritumoral routes ofadministration. A preferred manner of administering a separateanti-cancer agent is by targeting to a cancer cell. In this regard,examples of cancer-specific, cell-surface molecules include placentalalkaline phosphatase (testicular and ovarian cancer), pan carcinoma(small cell lung cancer), polymorphic epithelial mucin (ovarian cancer),prostate-specific membrane antigen, α-fetoprotein, B-lymphocyte surfaceantigen (B1-cell lymphoma), truncated EGFR (gliomas), idiotypes (B-celllymphoma), gp95/gp97 (melanoma), N-CAM (small cell lung carcinoma),cluster w4 (small cell lung carcinoma), cluster 5A (small cellcarcinoma), cluster 6 (small cell lung carcinoma), PLAP (seminomas,ovarian cancer, and non-small cell lung cancer), CA-125 (lung andovarian cancers), ESA (carcinoma), CD19, 22 or 37 (B-cell lymphoma), 250kD proteoglycan (melanoma), P55 (breast cancer), TCR-IgH fusion(childhood T-cell leukemia), blood group A antigen in B or 0 typeindividual (gastric and colon tumors), and the like. See, e.g., U.S.Pat. No. 6,080,725 for other examples.

Examples of cancer-specific, cell-surface receptors include erbB-2,erbB-3, erbB-4, IL-2 (lymphoma and leukemia), IL-4 (lymphoma andleukemia), IL-6 (lymphoma and leukemia), MSH (melanoma), transferrin(gliomas), tumor vasculature integrins, and the like. Preferredcancer-specific, cell-surface receptors include erbB-2 and tumorvasculature integrins, such as CD11a, CD11b, CD11c, CD18, CD29, CD51,CD61, CD66d, CD66e, CD106, and CDw145.

There are a number of antibodies to cancer-specific, cell-surfacemolecules and receptors that are known. C46 Ab (Amersham) and 85A12 Ab(Unipath) to carcino-embryonic antigen, H17E2 Ab (ICRF) to placentalalkaline phosphatase, NR-LU-10 Ab NeoRx Corp.) to pan carcinoma, HMFC1Ab (ICRF) to polymorphic epithelial mucin, W14 Ab to B-human chorionicgonadotropin, RFB4 Ab (Royal Free Hospital) to B-lymphocyte surfaceantigen, A33 Ab (Genex) to human colon carcinoma, TA-99 Ab (Genex) tohuman melanoma, antibodies to c-erbB2 (JP 7309780, JP 8176200 and JP7059588), and the like. ScAbs can be developed, based on suchantibodies, using techniques known in the art (see for example, Bind etal., Science 242: 423-426 (1988), and Whitlow et al., Methods 2(2):97-105 (1991)).

Generally, when Tey 1 (or a conjugate or fusion protein thereof) isadministered to an animal, such as a mammal, in particular a human, itis desirable that Tey 1 be administered in a dose of from about 1 toabout 1,000 μg/kg body weight/treatment when given parenterally. Higheror lower doses may be chosen in appropriate circumstances. For instance,the actual dose and schedule can vary depending on whether thecomposition is administered in combination with other pharmaceuticalcompositions, or depending on interindividual differences inpharmacokinetics, drug disposition, and metabolism. One skilled in theart easily can make any necessary adjustments in accordance with thenecessities of the particular situation.

Those of ordinary skill in the art can easily make a determination ofthe amount of an above-described isolated and purified nucleic acidmolecule to be administered to an animal, such as a mammal, inparticular a human. The dosage will depend upon the particular method ofadministration, including any vector or promoter utilized. For purposesof considering the dose in terms of particle units (pu), also referredto as viral particles, it can be assumed that there are 100particles/pfu (e.g., 1×10¹² pfu is equivalent to 1×10¹⁴ pu). An amountof recombinant virus, recombinant DNA vector or RNA genome sufficient toachieve a tissue concentration of about 10² to about 10¹² particles perml is preferred, especially of about 10⁶ to about 10¹⁰ particles per ml.In certain applications, multiple daily doses are preferred. Moreover,the number of doses will vary depending on the means of delivery and theparticular recombinant virus, recombinant DNA vector or RNA genomeadministered.

A targeting moiety also can be used in the contact of a cell with anabove-described isolated or purified nucleic acid molecule. In thisregard, any molecule that can be linked with the therapeutic nucleicacid directly or indirectly, such as through a suitable deliveryvehicle, such that the targeting moiety binds to a cell-surfacereceptor, can be used. The targeting moiety can bind to a cell through areceptor, a substrate, an antigenic determinant or another binding siteon the surface of the cell. Examples of a targeting moiety include anantibody (i.e., a polyclonal or a monoclonal antibody), animmunologically reactive fragment of an antibody, an engineeredimmunoprotein and the like, a protein (target is receptor, as substrate,or regulatory site on DNA or RNA), a polypeptide (target is receptor), apeptide (target is receptor), a nucleic acid, which is DNA or RNA (i.e.,single-stranded or double-stranded, synthetic or isolated and purifiedfrom nature; target is complementary nucleic acid), a steroid (target issteroid receptor), and the like. Analogs of targeting moieties thatretain the ability to bind to a defined target also can be used. Inaddition, synthetic targeting moieties can be designed, such as to fit aparticular epitope. Alternatively, the therapeutic nucleic acid can beencapsulated in a liposome comprising on its surface the targetingmoiety.

The targeting moiety includes any linking group that can be used to joina targeting moiety to, in the context of the present invention, anabove-described nucleic acid molecule. It will be evident to one skilledin the art that a variety of linking groups, including bifunctionalreagents, can be used. The targeting moiety can be linked to thetherapeutic nucleic acid by covalent or non-covalent bonding. If bondingis non-covalent, the conjugation can be through hydrogen bonding, ionicbonding, hydrophobic or van der Waals interactions, or any otherappropriate type of binding.

Also in view of the above, the present invention provides a method ofdiagnosing cancer in a mammal. The method comprises (a) obtaining a testsample from the mammal, and (b) assaying the test sample for the levelof Tey 1. A decrease in the level of Tey 1 in the test sample ascompared to the level of Tey 1 in a control sample is diagnostic for thecancer.

The test sample used in conjunction with the invention can be any ofthose typically used in the art. For example, the test sample can betissue. Typically, the tissue is metastatic (e.g., cancerous) and isobtained by means of a biopsy. Such tissue can include bone marrow,lymph nodes, skin, and any organ that may develop cancerous cells.Preferably, however, the test sample is taken from a source in whichsecreted proteins will be most prevalent. Accordingly, the test sampleis preferably serum, wherein the serum is obtained from methods known inthe art, such as a blood sample.

A number of assays are contemplated for use in the present inventivemethods of diagnosing cancer. A number of these assays are described inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1989. Microarrays, suchas those described in U.S. Pat. Nos. 6,197,506 and 6,040,138, also canbe used to detect and quantify Tey 1. It will be understood that thetype of assay used will depend on whether DNA, RNA or a protein (or apolypeptide thereof) is being assayed.

As used herein, the term “increased level” can be defined as detectingTey 1 in a mammal at a level above that which is considered normal. Forexample, the level of Tey 1 in a test sample is increased when the copynumber of the gene encoding the Tey 1 is greater than 1, the mRNAencoding Tey 1 is about 0.001-1%, or Tey 1 (or a polypeptide thereof) isdetected in an amount of about 1-10,000 ng/ml.

When a nucleic acid (i.e., DNA or RNA) is assayed, various assays can beused to measure the presence and/or level of nucleic acid present. Forexample, when only the detection of Tey 1 is necessary to diagnoseeffectively the cancer, assays including PCR and microarray analysis canbe used. In certain embodiments, it will be necessary to detect thequantity of Tey 1 present. In these embodiments, it will be advantageousto use various hybridization techniques known in the art that caneffectively measure the level of Tey 1 in a test sample. When the Tey 1comprises DNA, such hybridization techniques can include, for example,Southern hybridization (i.e., a Southern blot), in situ hybridizationand microarray analysis. Similarly, when the Tey 1 comprises RNA,Northern hybridization (i.e., a Northern blot), in situ hybridizationand microarray analysis are contemplated.

It will be understood that, in such assays, a nucleic acid sequence thatspecifically binds to or associates with a nucleic acid encoding Tey 1,whether DNA or RNA, can be attached to a label for determininghybridization. A wide variety of appropriate labels are known in theart, including fluorescent, radioactive, and enzymatic labels as well asligands, such as avidin/biotin, which are capable of being detected.Preferably, a fluorescent label or an enzyme tag, such as urease,alkaline phosphatase or peroxidase, is used instead of a radioactive orother environmentally undesirable label. In the case of enzyme tags,colorimetric indicator substrates are known which can be employed toprovide a detection means visible to the human eye orspectrophotometrically to identify specific hybridization withcomplementary Tey 1 nucleic acid-containing samples.

When a nucleic acid encoding the Tey 1 is amplified in the context of adiagnostic application, the nucleic acid used as a template foramplification is isolated from cells contained in the test sample,according to standard methodologies. (Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989). The nucleic acid can be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it can be desirableto convert the RNA to cDNA.

In a typical amplification procedure, pairs of primers that selectivelyhybridize to nucleic acids corresponding to Tey 1 are contacted with thenucleic acid under conditions that permit selective hybridization. Oncehybridized, the nucleic acid-primer complex is contacted with one ormore enzymes that facilitate template-dependent nucleic acid synthesis.Multiple rounds of amplification, also referred to as “cycles,” areconducted until a sufficient amount of amplification product isproduced.

Various template-dependent processes are available to amplify the Tey 1present in a given test sample. As with the various assays, a number ofthese processes are described in Sambrook et al. (1989), supra. One ofthe best-known amplification methods is the polymerase chain reaction(PCR). Similarly, a reverse transcriptase PCR (RT-PCR) can be used whenit is desired to convert mRNA into cDNA. Alternative methods for reversetranscription utilize thermostable DNA polymerases and are described inWO 90/07641, for example.

Other methods for amplification include the ligase chain reaction (LCR),which is disclosed in U.S. Pat. No. 4,883,750; isothermal amplification,in which restriction endonucleases and ligases are used to achieve theamplification of target molecules that contain nucleotide5′-[alpha-thio]-triphosphates in one strand (Walker et al., Proc. Natl.Acad. Sci. USA 89: 392-396 (1992)); strand displacement amplification(SDA), which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation; and repair chain reaction (RCR),which involves annealing several probes throughout a region targeted foramplification, followed by a repair reaction in which only two of thefour bases are present. The other two bases can be added as biotinylatedderivatives for easy detection. Target-specific sequences also can bedetected using a cyclic probe reaction (CPR). In CPR, a probe having 3′and 5′ sequences of non-specific DNA and a middle sequence of specificRNA is hybridized to DNA, which is present in a sample. Uponhybridization, the reaction is treated with RNase H, and the products ofthe probe are identified as distinctive products, which are releasedafter digestion. The original template is annealed to another cyclingprobe and the reaction is repeated. A number of other amplificationprocesses are contemplated; however, the invention is not limited as towhich method is used.

Following amplification of the Tey 1, it can be desirable to separatethe amplification product from the template and the excess primer forthe purpose of determining whether specific amplification has occurred.In one embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al. (1989), supra.

Alternatively, chromatographic techniques can be employed to effectseparation. There are many kinds of chromatography which can be used inthe context of the present inventive methods e.g., adsorption,partition, ion-exchange and molecular sieve, and many specializedtechniques for using them including column, paper, thin-layer and gaschromatography (Freifelder, Physical Biochemistry Applications toBiochemistry and Molecular Biology, 2nd Ed., Wm. Freeman and Co., NewYork, N.Y. (1982)).

Amplification products must be visualized in order to confirmamplification of the Tey 1 sequence. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified Tey 1 sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, where the other member of the binding paircarries a detectable moiety (i.e., a label).

One example of the foregoing is described in U.S. Pat. No. 5,279,721,which discloses an apparatus and method for the automatedelectrophoresis and transfer of nucleic acids. The apparatus permitselectrophoresis and blotting without external manipulation of the geland is ideally suited to carrying out methods according to the presentinvention.

It will be understood that the probes described above are limited in asmuch as any nucleic-acid sequence can be used as long as the nucleicacid sequence is hybridizable to nucleic acids encoding Tey 1 orfunctional sequence analogs thereof. For example, a nucleic acid ofpartial sequence can be used to quantify the expression of astructurally related gene or the full-length genomic or cDNA clone fromwhich it is derived.

Preferably, the hybridization is done under high stringency conditions.By “high stringency conditions” is meant that the probe specificallyhybridizes to a target sequence in an amount that is detectably strongerthan non-specific hybridization. High stringency conditions, then, wouldbe conditions which would distinguish a polynucleotide with an exactcomplementary sequence, or one containing only a few scatteredmismatches from a random sequence that happened to have a few smallregions (e.g., 3-10 bases) that matched the probe. Such small regions ofcomplementarity, are more easily melted than a full-length complement of14-17 or more bases and high stringency hybridization makes them easilydistinguishable. Relatively high stringency conditions would include,for example, low salt and/or high temperature conditions, such asprovided by about 0.02-0.1 M NaCl or the equivalent, at temperatures ofabout 50-70° C. Such high stringency conditions tolerate little, if any,mismatch between the probe and the template or target strand, and areparticularly suitable for detecting expression of specific Tey 1. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

When a Tey 1 protein or polypeptide fragment thereof is assayed, variousassays (i.e., immunobinding assays) are contemplated to detect ormeasure the quantity of Tey 1. In such embodiments, the Tey 1 can beemployed to detect antibodies having reactivity therewith, or,alternatively, antibodies can be prepared and employed to detect theTey 1. The steps of various useful immunodetection assays have beendescribed in Nakamura et al., Handbook of Experimental Immunology (4thEd.), Wol. 1, Chapter 27, Blackwell Scientific Publ., Oxford (1987);Nakamura et al., Enzyme Immunoassays: Heterogenous and HomogenousSystems, Chapter 27 (1987) and include Western hybridization (i.e.,Western blots), immunoaffinity purification, immunoaffinity detection,enzyme-linked immunosorbent assay (e.g., an ELISA), andradioimmunoassay. A microarray also can be used to detect or measure thelevels of Tey 1.

In general, the immunobinding assays involve obtaining a test samplesuspected of containing a protein, peptide or antibody corresponding toa Tey 1, and contacting the sample with an antibody in accordance withthe present invention, as the case may be, under conditions effective toallow the formation of immunocomplexes. Indeed, a mammal can bediagnosed with a cancer by either detecting Tey 1 or an antibody thatrecognizes Tey 1, or by quantifying the levels of Tey 1 or an antibodythat recognizes Tey 1. Any suitable antibody can be used in conjunctionwith the present invention such that the antibody is specific for Tey 1.

The immunobinding assays for use in the present invention includemethods for detecting or quantifying the amount of Tey 1 in a sample,which methods require the detection or quantitation of any immunecomplexes formed during the binding process. Here, a test samplesuspected of containing a Tey 1 would be obtained from a mammal andsubsequently contacted with an antibody (e.g., MHS-5). The detection orthe quantification of the amount of immune complexes formed under thespecific conditions is then performed.

Contacting the biological sample with an antibody that recognizes a Tey1 under conditions effective and for a period of time sufficient toallow formation of immune complexes (primary immune complexes) isgenerally a matter of simply adding the antibody to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, anyantigens. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or Western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well-known inthe art and can be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. Patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837,3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Ofcourse, additional advantages can be realized by using a secondarybinding ligand, such as a second antibody or a biotin/avidin ligandbinding arrangement, as is known in the art.

The antibody which is used in the context of the present invention can,itself, be linked to a detectable label, wherein one would then simplydetect this label, thereby allowing the presence of or the amount of theprimary immune complexes to be determined.

Alternatively, the first added component that becomes bound within theprimary immune complexes can be detected by means of a second bindingligand that has binding affinity for the first antibody. In these cases,the second binding ligand is, itself, often an antibody, which can betermed a “secondary” antibody. The primary immune complexes arecontacted with the labeled, secondary binding ligand, or antibody, underconditions effective and for a period of time sufficient to allow theformation of secondary immune complexes. The secondary immune complexesare then washed to remove any non-specifically bound labeled secondaryantibodies or ligands, and the remaining label in the secondary immunecomplexes is then detected.

Further methods include the detection of primary immune complexes by atwo-step approach. A second binding ligand, such as an antibody, thathas binding affinity for the first antibody is used to form secondaryimmune complexes, as described above. After washing, the secondaryimmune complexes are contacted with a third binding ligand or antibodythat has binding affinity for the second antibody, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed.

It will be understood that other diagnostic tests can be used inconjunction with the diagnostic tests described herein to enhancefurther the accuracy of diagnosing a mammal with a cancer. For example,a monoclonal antibody, such as the ones described in U.S. Pat. No.4,569,788, can be used effectively in diagnosing small-cell lung cancerover non small-cell lung cancer.

A method of prognosticating cancer in a mammal is also provided. Themethod comprises (a) obtaining a test sample from the mammal, and (b)assaying the test sample for the level of Tey 1. The level of Tey 1 inthe test sample can be measured by comparing the level of Tey 1 inanother test sample obtained from the mammal over time in accordancewith the methods described above. An increase in the level of Tey 1 overtime is indicative of a positive prognosis and a decrease in the levelof Tey 1 over time is indicative of a negative prognosis. The method canbe used to assess the efficacy of treatment of the cancer.

EXAMPLE

The following example serves to illustrate the present invention and isnot intended to limit its scope in any way.

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1997),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1998),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    3, Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Birren et al., Genome Analysis: A Laboratory Manual Series, Volume    4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring    Harbor, N.Y. (1999),-   Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y. (1988),-   Harlow et al., Using Antibodies: A Laboratory Manual, Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999),-   Hoffman, Cancer and the Search for Selective Biochemical Inhibitors,    CRC Press (1999),-   Pratt, The Anticancer Drugs, 2nd edition, Oxford University Press,    NY (1994), and-   Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd    edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y. (1989).

Example 1

This example describes the cloning and sequencing of the Tey 1.

A 60 Kb bacterial artificial chromosome (BAC) clone on human chromosome8, encoding a metastasis suppressor gene for highly metastatic ratprostate cancer cell line AT6.3, was identified (Nihei et al., CancerResearch (2001), accepted). Several techniques were used to identifygenes in this region, including cDNA selection (Morgan et al., NucleicAcids Research 20: 5173-5179 (1992)) and genomic sequencing of the BACclone (Platzer et al., Genome Research 7: 592-605 (1997)).

cDNA selection was performed as described (Morgan et al. (1992), supra).Poly (A)+ RNA was isolated from a microcell hybrid (AT6.3-8-22) whichcontained the metastasis suppressor gene (Sambrook et al. (1989), supra)using a poly(A) pure mRNA isolation kit (Ambion, Austin, Tex.). Normalhuman prostate poly (A)+ RNA (Clontech, Palo Alto, Calif.) was also usedseparately. Random-primed double stranded cDNA was synthesized from 5 μgof these RNAs using SuperScript Choice System for cDNA Synthesis (LifeTechnologies, Grand Island, N.Y.), digested with Sau 3 μl, ligated toMbo linker I, and amplified by PCR as described (Sambrook et al.,(1989), supra). BAC DNA was isolated using NucleoBond BAC maxi kit(Clontech), digested with Sau 3A1, ligated to Mbo linker II, andamplified by PCR with 5′ biotinylated primer as described (Morgan et al.(1992), supra). After purification and size selection by PCRpurification kit (Qiagen, Valencia, Calif.), repeat sequences in boththe biontin-labeled selector DNA and the target cDNA were suppressed byannealing with CotI DNA (Life Technologies). the selector BAC DNA (100ng) and the target cDNA (1 μg) were hybridized at 65° C. in a 100 μlsolution of 6×SSC and 0.1% SDS for 16 hr. Selected cDNAs were capturedwith streptavidin-cojugated paramagnetic particles (Promega, Madison,WI, eluted in 50 μl of 50 mM NaOH, and amplified by PCR as described(Morgan et al. (1992), supra). These amplified eluted cDNAs wererecycled through the above process for secondary selection. Second-roundselected cDNAs were digested with Eco RI, and ligated into EcoRI-digested and phosphatased pUC18 plasmid vector. These cloned cDNAinserts were then transformed into XL-10 gold ultracompetent cells(Stratagene, La Jolla, Calif.). Colony PCR was performed on 400 whitecolonies with M13/pUC sequencing primers (Life Technologies), and thePCR products were blotted on Hybond N+nylon membranes (AmershamPharmacia, Piscataway, N.J.). These blots were hybridized with the 60 KbBAC DNA and no insert BAC DNA by standard methods (Sambrook et al.(1989), supra). Approximately 100 clones were mapped back to the 60 KbBAC clone and sequenced using dRhodamine cycle sequencing kit (AppliedBiosystems, Foster City, Calif.) on an ABI 377 automated sequencer(Applied Biosytems). Sequences of transcripts obtained by cDNA selectionwere verified against the genomic sequence of the 60 Kb BAC clone.

Genomic sequencing of the 60 Kb BAC clone was performed by the shotgunmethod (Platzer et al., Genome Research 7: 592-605 (1997)) at the NIHIntramural Sequencing Center. The genomic sequence information was usedto identify expressed sequence tags (ESTs) using the BLAST program(Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), and to performgene screening using GRAIL and GENSCAN gene prediction programs(Uberbacher et al., PNAS USA 88: 11261-11265 (1991)).

These analyses identified a single novel gene within this region. Inorder to isolate a full-length cDNA, three different cDNA libraryscreening methods were used. Traditional plaque filter hybridizationwith an adult human prostate cDNA library (Clontech) and kidney cDNAlibrary (Stratagene) was performed by standard methods (Altschul et al.(1990), supra). A cDNA library using poly(A)+ RNA isolated from theAT6.3 rat prostate cancer cells transfected with the 60 Kb BAC clone(SuperScript Choice System for cDNA Synthesis, Life Technologies) alsowas constructed and used for the third screening. The accuracy of thecDNA sequences was confirmed by RT-PCR and Northern blot analysis usinggene-specific primer pairs and probes by standard methods (Altschul etal. (1990), supra).

Direct comparison of the genomic sequence of the 60 Kb BAC clone and thecDNA sequences allowed the exon-intron boundaries to be determined forall exons.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

1. An isolated or purified nucleic acid molecule consisting essentiallyof a nucleotide sequence encoding the metastasis suppressor gene locatedat p21-p12 on chromosome 8 of a human (Tey 1) or a fragment thereofcomprising at least 455 contiguous nucleotides.
 2. The isolated orpurified nucleic acid molecule of claim 1, which (i) encodes the aminoacid sequence of SEQ ID NO: 3 or 5, (ii) consists essentially of thenucleotide sequence of SEQ ID NO: 1, 2 or 4 or a fragment thereofcomprising at least 455 contiguous nucleotides, (iii) hybridizes underlow stringency conditions to an isolated or purified nucleic acidmolecule consisting essentially of the nucleotide sequence that iscomplementary to SEQ ID NO: 1, 2 or 4 or a fragment thereof comprisingat least 455 contiguous nucleotides, or (iv) shares 50% or more identitywith SEQ ID NO: 1, 2 or
 4. 3. An isolated or purified nucleic acidmolecule consisting essentially of a nucleotide sequence encoding avariant Tey 1, which comprises one or more insertions, deletions,substitutions, and/or inversions, wherein the variant Tey 1 encoded bythe isolated or purified nucleic acid molecule does not differfunctionally from the corresponding unmodified Tey 1, or a fragmentthereof comprising at least 455 contiguous nucleotides.
 4. The isolatedor purified nucleic acid molecule of claim 3, wherein the variant Tey 1is able to suppress metastasis of a highly metastatic prostatic tumorcell line in vivo at least about 90% as well as the unmodified Tey 1comprising SEQ ID NO: 3 or
 5. 5. An isolated or purified nucleic acidmolecule consisting essentially of a nucleotide sequence that iscomplementary to a nucleotide sequence encoding Tey 1 or a fragmentthereof comprising at least 455 contiguous nucleotides.
 6. The isolatedor purified nucleic acid molecule of claim 5, which (i) is complementaryto a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 or 5, (ii) is complementary to the nucleotide sequence of SEQ ID NO:1, 2 or 4 or a fragment thereof comprising at least 455 contiguousnucleotides, (iii) hybridizes under low stringency conditions to anisolated or purified nucleic acid molecule consisting essentially of SEQID NO: 1, 2 or 4 or a fragment thereof comprising at least 455contiguous nucleotides, or (iv) shares 50% or more identity with thenucleotide sequence that is complementary to SEQ ID NO: 1, 2 or
 4. 7. Anisolated or purified nucleic acid molecule consisting essentially of anucleotide sequence that is complementary to a nucleotide sequenceencoding a variant Tey
 1. 8. A vector comprising the isolated orpurified nucleic acid molecule of claim 1, optionally as part of anencoded fusion protein.
 9. A vector comprising the isolated or purifiednucleic acid molecule of claim 2, optionally as part of an encodedfusion protein.
 10. A vector comprising the isolated or purified nucleicacid molecule of claim 3, optionally as part of an encoded fusionprotein.
 11. A vector comprising the isolated or purified nucleic acidmolecule of claim 4, optionally as part of an encoded fusion protein.12. A vector comprising the isolated or purified nucleic acid moleculeof claim
 5. 13. A vector comprising the isolated or purified nucleicacid molecule of claim
 6. 14. A vector comprising the isolated orpurified nucleic acid molecule of claim
 7. 15. A cell comprising andexpressing the isolated or purified nucleic acid molecule of claim 1,optionally in the form of a vector.
 16. A cell comprising and expressingthe isolated or purified nucleic acid molecule of claim 2, optionally inthe form of a vector.
 17. A cell comprising and expressing the isolatedor purified nucleic acid molecule of claim 3, optionally in the form ofa vector.
 18. A cell comprising and expressing the isolated or purifiednucleic acid molecule of claim 4, optionally in the form of a vector.19. A cell comprising and expressing the isolated or purified nucleicacid molecule of claim 5, optionally in the form of a vector.
 20. A cellcomprising and expressing the isolated or purified nucleic acid moleculeof claim 6, optionally in the form of a vector.
 21. A cell comprisingand expressing the isolated or purified nucleic acid molecule of claim7, optionally in the form of a vector.
 22. An isolated or purifiedpolypeptide molecule consisting essentially of an amino acid sequenceencoding Tey 1 or at least 6 contiguous amino acids of Tey 1, which isoptionally glycosylated, amidated, carboxylated, phosphorylated,esterified, N-acylated or converted into an acid addition salt.
 23. Aconjugate or fusion protein comprising the isolated or purifiedpolypeptide molecule of claim 22 and a therapeutically orprophylactically active agent.
 24. The conjugate of claim 23, whereinthe therapeutically or prophylactically active agent is an anti-canceragent.
 25. A composition comprising the isolated or purified polypeptidemolecule of claim 22, optionally in the form of a conjugate or a fusionprotein comprising a therapeutically or prophylactically active agent,and an excipient or an adjuvant.
 26. An isolated or purified polypeptidemolecule consisting essentially of an amino acid sequence encoding avariant Tey 1 or at least 6 contiguous amino acids of a variant Tey 1,which is optionally glycosylated, amidated, carboxylated,phosphorylated, esterified N-acylated or converted into an acid additionsalt.
 27. A conjugate or fusion protein comprising the isolated orpurified polypeptide molecule of claim 26 and a therapeutically orprophylactically active agent.
 28. The conjugate of claim 27, whereinthe therapeutically or prophylactically active agent is an anti-canceragent.
 29. A composition comprising the isolated or purified polypeptidemolecule of claim 26, optionally in the form of a conjugate or a fusionprotein comprising a therapeutically or prophylactically active agent,and an excipient or an adjuvant.
 30. A method of treating cancerprophylactically or therapeutically in a mammal, which method comprisesadministering to the mammal an effective amount of: (a) an isolated orpurified nucleic acid molecule encoding Tey 1, optionally in the form ofa vector, or (b) an isolated or purified Tey 1 polypeptide, optionallyin the form of a conjugate or fusion protein, whereupon the mammal istreated for the cancer prophylactically or therapeutically.
 31. Themethod of claim 30, wherein the cancer is prostate cancer.
 32. A methodof diagnosing cancer in a mammal, which method comprises: (a) obtaininga test sample from the mammal, and (b) assaying the test sample for thelevel of Tey 1, wherein a decrease in the level of Tey 1 in the testsample as compared to the level of Tey 1 in a control sample isdiagnostic for the cancer.
 33. A method of prognosticating cancer in amammal, which method comprises: (a) obtaining a test sample from themammal, and (b) assaying the test sample for the level of Tey 1, whereinan increase in the level of Tey 1 over time is indicative of a positiveprognosis and a decrease in the level of Tey 1 over time is indicativeof a negative prognosis.
 34. The method of claim 33, wherein the methodis used to assess the efficacy of treatment of the cancer.